The goal of Project 0003 is to discover the genes that contribute to isolated cleft lip and palate, a common disorder with complex etiology. This project will study IRF6, a gene that encodes the transcription factor Interferon Regulatory Factor 6. Mutations in IRF6 cause Van der Woude syndrome (VWS). VWS is an outstanding clinical model for isolated cleft lip and palate because the phenotypes of VWS and isolated cleft lip and palate are similar. It is also an outstanding genetic model, since alleles of IRF6 are highly associated with isolated cleft lip and palate in nine geographically distinct populations. These results prove that IRF6 is a key component of a critical pathway in palate development. In this proposal we use mouse models to identify the IRF6 pathway and other genes therein. There are two main hypotheses, 1) IRF6 is a mediator of Tgfb signaling and 2) IRF6 gene targets are essential for palate development. There are three Specific Aims. Specific Aim 1 will determine which cell-type expresses Irf6 in the palate. This basic information will immediately suggest Irf6 pathways and gene targets. Specific Aim 2 will determine which pathways require Irf6 for palate development. The Tgfb3 signaling pathway is essential for palate development and Tgfb3 is expressed at about the same time and place as Irf6. Knockout mice will be used to test the hypotheses that Tgfb3 is essential for expression of Irf6 in the palate shelves. Specific Aim 3 will use microarray analysis to identify the target genes for Irf6 during palate development. In addition, if Irf6 is a mediator for Tgfb3, then gene expression in the palate will be similar in Tgfb3-/- and Irf6-/- mice. Addressing these aims will advance our understanding of Irf6, a gene that is essential in craniofacial development. Further, the IRF6 pathways and targets, discovered in this proposal, will be assessed for their involvement in cleft lip and palate in Project 0001 and in palate development in Project 0004 and Cores 9003 and 9004. This aggregate knowledge will enhance our ability to determine the causes of, and develop preventive interventions for, orofacial clefts.